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https://www.readbyqxmd.com/read/28475612/calmodulin-like-proteins-localized-to-the-conoid-regulate-motility-and-cell-invasion-by-toxoplasma-gondii
#1
Shaojun Long, Kevin M Brown, Lisa L Drewry, Bryan Anthony, Isabelle Q H Phan, L David Sibley
Toxoplasma gondii contains an expanded number of calmodulin (CaM)-like proteins whose functions are poorly understood. Using a combination of CRISPR/Cas9-mediated gene editing and a plant-like auxin-induced degron (AID) system, we examined the roles of three apically localized CaMs. CaM1 and CaM2 were individually dispensable, but loss of both resulted in a synthetic lethal phenotype. CaM3 was refractory to deletion, suggesting it is essential. Consistent with this prediction auxin-induced degradation of CaM3 blocked growth...
May 5, 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28470616/in-vivo-biotinylation-of-antigens-in-e-coli
#2
Susanne Gräslund, Pavel Savitsky, Susanne Müller-Knapp
Site-specific biotinylation of proteins is often the method of choice to enable efficient immobilization of a protein on a surface without interfering with protein folding. The tight interaction of biotin and streptavidin is frequently used to immobilize an antigen during phage display selections of binders. Here we describe a method of in vivo biotinylation of proteins during expression in E. coli, by tagging the protein with the short biotin acceptor peptide sequence, Avi tag, and co-expression of the E. coli biotin ligase (BirA) resulting in precise biotinylation of a specific lysine residue in the tag...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28466579/a-conserved-regulatory-mechanism-in-bifunctional-biotin-protein-ligases
#3
Jingheng Wang, Dorothy Beckett
Class II bifunctional biotin protein ligases (BirA), which catalyze post-translational biotinylation and repress transcription initiation, are broadly distributed in eubacteria and archaea. However, it is unclear if these proteins all share the same molecular mechanism of transcription regulation. In Escherichia coli the corepressor biotinoyl-5'-AMP (bio-5'-AMP), which is also the intermediate in biotin transfer, promotes operator binding and resulting transcription repression by enhancing BirA dimerization...
May 2, 2017: Protein Science: a Publication of the Protein Society
https://www.readbyqxmd.com/read/28463988/translocation-of-the-papillomavirus-l2-vdna-complex-across-the-limiting-membrane-requires-the-onset-of-mitosis
#4
Christine M Calton, Matthew P Bronnimann, Ariana R Manson, Shuaizhi Li, Janice A Chapman, Marcela Suarez-Berumen, Tatum R Williamson, Sudheer K Molugu, Ricardo A Bernal, Samuel K Campos
The human papillomavirus type 16 (HPV16) L2 protein acts as a chaperone to ensure that the viral genome (vDNA) traffics from endosomes to the trans-Golgi network (TGN) and eventually the nucleus, where HPV replication occurs. En route to the nucleus, the L2/vDNA complex must translocate across limiting intracellular membranes. The details of this critical process remain poorly characterized. We have developed a system based on subcellular compartmentalization of the enzyme BirA and its cognate substrate to detect membrane translocation of L2-BirA from incoming virions...
May 2017: PLoS Pathogens
https://www.readbyqxmd.com/read/28454708/functional-characterisation-of-burkholderia-pseudomallei-biotin-protein-ligase-a-toolkit-for-anti-melioidosis-drug-development
#5
Thomas E H Bond, Alanna E Sorenson, Patrick M Schaeffer
Burkholderia pseudomallei (Bp) is the causative agent of melioidosis. The bacterium is responsible for 20% of community-acquired sepsis cases and 40% of sepsis-related mortalities in northeast Thailand, and is intrinsically resistant to aminoglycosides, macrolides, rifamycins, cephalosporins, and nonureidopenicillins. There is no vaccine and its diagnosis is problematic. Biotin protein ligase (BirA) which is essential for fatty acid synthesis has been proposed as a drug target in bacteria. Very few bacterial BirA have been characterized, and a better understanding of these enzymes is necessary to further assess their value as drug targets...
June 2017: Microbiological Research
https://www.readbyqxmd.com/read/28381627/characterization-of-rbp9-and-rbp10-two-developmentally-regulated-rna-binding-proteins-in-trypanosoma-brucei
#6
Luis Miguel De Pablos, Steve Kelly, Janaina de Freitas Nascimento, Jack Sunter, Mark Carrington
The fate of an mRNA is determined by its interaction with proteins and small RNAs within dynamic complexes called ribonucleoprotein complexes (mRNPs). In Trypanosoma brucei and related kinetoplastids, responses to internal and external signals are mainly mediated by post-transcriptional processes. Here, we used proximity-dependent biotin identification (BioID) combined with RNA-seq to investigate the changes resulting from ectopic expression of RBP10 and RBP9, two developmentally regulated RNA-binding proteins (RBPs)...
April 2017: Open Biology
https://www.readbyqxmd.com/read/28336960/co-expression-of-bira-with-biotin-bait-achieves-in-vivo-biotinylation-of-overexpressed-stable-n-glycosylated-srage-in-transgenic-silkworms
#7
Miyuki Kumano-Kuramochi, Ken-Ichiro Tatematsu, Mayumi Ohnishi-Kameyama, Mari Maeda-Yamamoto, Toshiro Kobori, Hideki Sezutsu, Sachiko Machida
Here, we demonstrated the expression of the N-glycosylated extracellular ligand binding domain of receptor for advanced glycation end products (sRAGE) in middle silk glands (MSGs) of transgenic silkworms using the GAL4/UAS system. Over 1 mg of sRAGE was obtained from one transgenic silkworm. sRAGE purified from the silkworm exhibited good stability and maintained specific ligand-binding ability. In addition, N-glycan analysis of sRAGE revealed that N-glucan completely lacked potentially allergenic fucose...
March 23, 2017: Scientific Reports
https://www.readbyqxmd.com/read/28331815/measuring-e-coli-and-bacteriophage-dna-in-cell-sonicates-to-evaluate-the-cal1-reaction-as-a-synthetic-biology-standard-for-qpcr
#8
Alexander Templar, Desmond M Schofield, Darren N Nesbeth
We measured the impact of the presence of total Escherichia coli (E. coli) cellular material on the performance of the Linear Regression of Efficiency (LRE) method of absolute quantitative PCR (LRE qPCR), which features the putatively universal CAL1 calibration reaction, which we propose as a synthetic biology standard. We firstly used a qPCR reaction in which a sequence present in the lone genomic BirA locus is amplified. Amplification efficiency for this reaction, a key metric for many quantitative qPCR methods, was inhibited by cellular material from bioreactor cultivation to a greater extent than material from shake flask cultivation...
March 2017: Biomolecular Detection and Quantification
https://www.readbyqxmd.com/read/28225217/proteomic-analysis-of-the-cullin4b-interactome-using-proximity-dependent-biotinylation-in-living-cells
#9
Hailong Zhang, Shupeng Li, Pingting Liu, Frankie H F Lee, Albert H C Wong, Fang Liu
Cullin 4B (CUL4B) mutations have been implicated in mental retardation and dopamine-related behaviors due to disruptions in their interaction with Cullin-RING E3 ligases (CRLs). Thus, further identification of CUL4B substrates can increase the knowledge of protein homeostasis and illuminate the role of CUL4B in neuropsychiatric disease. However, the transient nature of the coupling between CUL4B and its substrates is difficult to detect in vivo using current approaches, thus hampers efforts to investigate functions of CRLs within unperturbed living systems...
February 22, 2017: Proteomics
https://www.readbyqxmd.com/read/28212352/rta-occupancy-of-the-origin-of-lytic-replication-during-murine-gammaherpesvirus-68-reactivation-from-b-cell-latency
#10
Alexis L Santana, Darby G Oldenburg, Varvara Kirillov, Laraib Malik, Qiwen Dong, Roman Sinayev, Kenneth B Marcu, Douglas W White, Laurie T Krug
RTA, the viral Replication and Transcription Activator, is essential for rhadinovirus lytic gene expression upon de novo infection and reactivation from latency. Lipopolysaccharide (LPS)/toll-like receptor (TLR)4 engagement enhances rhadinovirus reactivation. We developed two new systems to examine the interaction of RTA with host NF-kappaB (NF-κB) signaling during murine gammaherpesvirus 68 (MHV68) infection: a latent B cell line (HE-RIT) inducible for RTA-Flag expression and virus reactivation; and a recombinant virus (MHV68-RTA-Bio) that enabled in vivo biotinylation of RTA in BirA transgenic mice...
February 16, 2017: Pathogens
https://www.readbyqxmd.com/read/28188527/parallel-exploration-of-interaction-space-by-bioid-and-affinity-purification-coupled-to-mass-spectrometry
#11
Geoffrey G Hesketh, Ji-Young Youn, Payman Samavarchi-Tehrani, Brian Raught, Anne-Claude Gingras
Complete understanding of cellular function requires knowledge of the composition and dynamics of protein interaction networks, the importance of which spans all molecular cell biology fields. Mass spectrometry-based proteomics approaches are instrumental in this process, with affinity purification coupled to mass spectrometry (AP-MS) now widely used for defining interaction landscapes. Traditional AP-MS methods are well suited to providing information regarding the temporal aspects of soluble protein-protein interactions, but the requirement to maintain protein-protein interactions during cell lysis and AP means that both weak-affinity interactions and spatial information is lost...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28129019/biochemical-analysis-of-force-sensitive-responses-using-a-large-scale-cell-stretch-device
#12
Derrick J Renner, Makena L Ewald, Timothy Kim, Soichiro Yamada
Physical force has emerged as a key regulator of tissue homeostasis, and plays an important role in embryogenesis, tissue regeneration, and disease progression. Currently, the details of protein interactions under elevated physical stress are largely missing, therefore, preventing the fundamental, molecular understanding of mechano-transduction. This is in part due to the difficulty isolating large quantities of cell lysates exposed to force-bearing conditions for biochemical analysis. We designed a simple, easy-to-fabricate, large-scale cell stretch device for the analysis of force-sensitive cell responses...
January 27, 2017: Cell Adhesion & Migration
https://www.readbyqxmd.com/read/28098257/a-comprehensive-platform-for-the-analysis-of-ubiquitin-like-protein-modifications-using-in-vivo-biotinylation
#13
Lucia Pirone, Wendy Xolalpa, Jón Otti Sigurðsson, Juanma Ramirez, Coralia Pérez, Monika González, Ainara Ruiz de Sabando, Félix Elortza, Manuel S Rodriguez, Ugo Mayor, Jesper V Olsen, Rosa Barrio, James D Sutherland
Post-translational modification by ubiquitin and ubiquitin-like proteins (UbLs) is fundamental for maintaining protein homeostasis. Efficient isolation of UbL conjugates is hampered by multiple factors, including cost and specificity of reagents, removal of UbLs by proteases, distinguishing UbL conjugates from interactors, and low quantities of modified substrates. Here we describe bioUbLs, a comprehensive set of tools for studying modifications in Drosophila and mammals, based on multicistronic expression and in vivo biotinylation using the E...
January 18, 2017: Scientific Reports
https://www.readbyqxmd.com/read/27876148/fc-specific-biotinylation-of-antibody-using-an-engineered-photoactivatable-z-biotin-and-its-biosensing-application
#14
Hong-Ming Yang, Ru-Meng Bao, Chang-Mei Yu, Yan-Na Lv, Wei-Fen Zhang, Jin-Bao Tang
The development of a site-specific and covalent attachment methodology is crucial for antibody-biotin conjugates to preserve the antigen-binding ability of antibodies and yield homogeneous products. In this study, an engineered photoactivatable Z-domain variant [an UV-active amino acid benzoylphenylalanine (Bpa) was genetically incorporated into the Z-domain] carrying one biotin molecule (ZBpa-Biotin) was prepared by employing aminoacyl-tRNA synthetase/suppressor tRNA and Avitag/BirA techniques. The site-specific and covalent attachment of IgG-biotin conjugates, viz...
January 1, 2017: Analytica Chimica Acta
https://www.readbyqxmd.com/read/27716942/an-assay-for-entry-of-secreted-fungal-effectors-into-plant-cells
#15
Libera Lo Presti, Bernd Zechmann, Jochen Kumlehn, Liang Liang, Daniel Lanver, Shigeyuki Tanaka, Ralph Bock, Regine Kahmann
Successful colonization of plants by prokaryotic and eukaryotic pathogens requires active effector-mediated suppression of defense responses and host tissue reprogramming. Secreted effector proteins can either display their activity in the apoplast or translocate into host cells and function therein. Although characterized in bacteria, the molecular mechanisms of effector delivery by fungal phytopathogens remain elusive. Here we report the establishment of an assay that is based on biotinylation of effectors in the host cytoplasm as hallmark of uptake...
January 2017: New Phytologist
https://www.readbyqxmd.com/read/27676121/determination-of-local-chromatin-composition-by-casid
#16
Elisabeth Schmidtmann, Tobias Anton, Pascaline Rombaut, Franz Herzog, Heinrich Leonhardt
Chromatin structure and function are determined by a plethora of proteins whose genome-wide distribution is typically assessed by immunoprecipitation (ChIP). Here, we developed a novel tool to investigate the local chromatin environment at specific DNA sequences. We combined the programmable DNA binding of dCas9 with the promiscuous biotin ligase BirA* (CasID) to biotinylate proteins in the direct vicinity of specific loci. Subsequent streptavidin-mediated precipitation and mass spectrometry identified both known and previously unknown chromatin factors associated with repetitive telomeric, major satellite and minor satellite DNA...
September 2, 2016: Nucleus
https://www.readbyqxmd.com/read/27615514/proximity-labeling-reveals-molecular-determinants-of-fgfr4-endosomal-transport
#17
Ellen Margrethe Haugsten, Vigdis Sørensen, Michaela Kunova Bosakova, Gustavo Antonio de Souza, Pavel Krejci, Antoni Wiedlocha, Jørgen Wesche
The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells...
October 7, 2016: Journal of Proteome Research
https://www.readbyqxmd.com/read/27506200/a-tissue-specific-protein-purification-approach-in-caenorhabditis-elegans-identifies-novel-interaction-partners-of-dlg-1-discs-large
#18
Selma Waaijers, Javier Muñoz, Christian Berends, João J Ramalho, Soenita S Goerdayal, Teck Y Low, Adja D Zoumaro-Djayoon, Michael Hoffmann, Thijs Koorman, Roderick P Tas, Martin Harterink, Stefanie Seelk, Jana Kerver, Casper C Hoogenraad, Olaf Bossinger, Baris Tursun, Sander van den Heuvel, Albert J R Heck, Mike Boxem
BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide...
2016: BMC Biology
https://www.readbyqxmd.com/read/27486190/in-vivo-biotinylation-of-the-toxoplasma-parasitophorous-vacuole-reveals-novel-dense-granule-proteins-important-for-parasite-growth-and-pathogenesis
#19
Santhosh M Nadipuram, Elliot W Kim, Ajay A Vashisht, Andrew H Lin, Hannah N Bell, Isabelle Coppens, James A Wohlschlegel, Peter J Bradley
UNLABELLED: Toxoplasma gondii is an obligate intracellular parasite that invades host cells and replicates within a unique parasitophorous vacuole. To maintain this intracellular niche, the parasite secretes an array of dense granule proteins (GRAs) into the nascent parasitophorous vacuole. These GRAs are believed to play key roles in vacuolar remodeling, nutrient uptake, and immune evasion while the parasite is replicating within the host cell. Despite the central role of GRAs in the Toxoplasma life cycle, only a subset of these proteins have been identified, and many of their roles have not been fully elucidated...
August 2, 2016: MBio
https://www.readbyqxmd.com/read/27445042/the-staphylococcus-aureus-group-ii-biotin-protein-ligase-bira-is-an-effective-regulator-of-biotin-operon-transcription-and-requires-the-dna-binding-domain-for-full-enzymatic-activity
#20
Sarah K Henke, John E Cronan
Group II biotin protein ligases (BPLs) are characterized by the presence of an N-terminal DNA binding domain that functions in transcriptional regulation of the genes of biotin biosynthesis and transport. The Staphylococcus aureus Group II BPL which is called BirA has been reported to bind an imperfect inverted repeat located upstream of the biotin synthesis operon. DNA binding by other Group II BPLs requires dimerization of the protein which is triggered by synthesis of biotinoyl-AMP (biotinoyl-adenylate), the intermediate in the ligation of biotin to its cognate target proteins...
November 2016: Molecular Microbiology
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